Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein

Finkler, Marc; Ravanbodshirazi, Sina; Hartz, Philip; Ott, Albrecht

doi:10.1016/j.bmc.2021.116207

Cited by 3 publications

(3 citation statements)

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“…The in vitro system described here presents the advantage of quick testing and prototyping of modified Arch-3, since only the DNA needs to be adapted. Moreover, even non-canonical amino acids can be incorporated [ 28 , 44 , 45 ]. Because G protein-coupled receptors involved in many signaling cascades exhibit a similar structure, we expect our work to be helpful for in vitro studies focusing on this kind of protein.…”

Section: Discussionmentioning

confidence: 99%

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Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Khangholi

Finkler

Seemann

et al. 2021

IJMS

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Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

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Section: Discussionmentioning

confidence: 99%

“…A cell-free expression system was used for the production and reconstitution of Arch-3-EGFP. Such in vitro systems enable better control of various biochemical parameters and processes than in vivo systems [ 25 , 26 , 27 , 28 ]. They are regularly used to study gene circuits or reaction cascades [ 29 , 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Khangholi

Finkler

Seemann

et al. 2021

IJMS

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“…Synthetic biology has applications that span from genomics, , transcriptomics, and proteomics , to metabolomics. , Examples are expression systems engineered to expand metabolic functions by including noncanonical amino acids into model proteins, − opening new directions in medicine. Synthetic oscillators, boolean logic gates, , and switches constitute examples of functional modules that produce logical operations at the transcriptional level.…”

Section: Introductionmentioning

confidence: 99%

A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA Reconstituted in an All E. coli Cell-Free Expression System

et al. 2021

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Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable ‘ON phase’ where the corresponding pap genes are expressed and a stable ‘OFF phase’ where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.

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Protocol to identify amino acids bound to tRNA by aminoacylation using mass spectrometry

Finkler,

Brandt,

Boutfol

et al. 2023

STAR Protocols

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Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein

Cited by 3 publications

References 44 publications

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA Reconstituted in an All E. coli Cell-Free Expression System

Protocol to identify amino acids bound to tRNA by aminoacylation using mass spectrometry

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